The acyl-CoA dehydrogenases (ACDs) are a family of evolutionarily related enzymes involved in the first step of the beta-oxidation of fatty acids and in the intermediate metabolism of leucine, isoleucine and valine. Deficiencies of these enzymes are increasingly being recognized as important causes of inherited defects of metabolism in humans. The long range objective of this application is to investigate important structure/ function relationships in the ACD gene family at the molecular level, and relate this information to mutations responsible for clinical deficiencies of these enzymes. Specific aims of this proposal include 1) analysis of the pathway of maturation of isovaleryl-CoA dehydrogenase (IVD), a member of this gene family, focusing on the process of tetramerization, 2) expression analysis of mutant IVD alleles from patients with deficiencies of IVD as a source of naturally occurring mutations of functional significance, and 3) characterization of structure/function relationships important in tetramerization of IVD and determination of substrate specificity of other ACD family members. The pathway of IVD maturation will be studied by metabolic labeling of IVD overexpressed in tissue culture cells via a eukaryotic expression vector, and by in vitro mitochondrial processing experiments. Cell fractionation studies will be used to identify the subcellular localization of this process, and gel filtration of mitochondrial extracts from in vitro transport experiments will identify intra-mitochondrial intermediates. In vitro mutagenesis will be used to produce IVD mutants deficient in tetramerization in order to identify amino acid motifs and residues critical to this process. Mutant alleles from patients with deficiencies of IVD will be studied via these same techniques in order to characterize the functional nature of the abnormal enzyme proteins produced by these patients' cells. Finally, structure/function relationships within this gene family will be dissected in a more rigorous fashion using the techniques of PCR in vitro site- specific mutagenesis and domain switching. These investigations will focus on the nature of the amino acid residues controlling the determination of substrate specificity of the various ACDs. Normal short and long chain acyl-CoA dehydrogenases will be expressed in a prokaryotic expression systems, and SCAD tertiary and quarternery structure will be studied by x- ray crystallography. Domain switching techniques will be used to synthesize hybrid ACDs with altered substrate binding specificities, and the substrate specificity will be correlated to the ACD sequences present to identify amino acid sequences important in determining this characteristic. These studies will lead to a more complete understanding of the ACD gene family, and an improvement in the ability to diagnose and treat patients with these disorders.